title: A kinetic test characterizes kinase intramolecular and intermolecular autophosphorylation mechanisms.
abstract: Many protein kinases catalyze their own activation by autophosphorylation. The mechanism of this is generally considered to be intermolecular and similar to that used in substrate phosphorylation. We derived the kinetic signatures of the four simplest autophosphorylation reactions and developed a test to determine the autoactivation mechanism of individual kinases. Whereas autophosphorylation of Nek7 and Plk4 occurred through an intermolecular mechanism, the kinases Aurora-A and Chk2 followed an intramolecular mechanism. Autophosphorylation of Aurora-A was accelerated in the presence of its protein activator TPX2. Nek9, the binding partner for Nek7, had a concentration-dependent effect such that low amounts enhanced autoactivation of Nek7 and high amounts were inhibitory. A structural model of Aurora-A undergoing autophosphorylation confirmed that an intramolecular mechanism is physically possible, and provided an explanation for how TPX2 could stimulate both autophosphorylation and substrate phosphorylation. The distinct mechanisms of autoactivation have consequences for cellular regulation because each molecule of a kinase that undergoes intramolecular autophosphorylation is activated individually, whereas the activity of kinases that undergo intermolecular autophosphorylation can be rapidly self-amplified in the cell. Local control of individual molecules, such as Aurora-A, may be particularly advantageous for a kinase with multiple, distinct cellular roles.
authors.affiliation: Division of Structural Biology, The Institute of Cancer Research, 237 Fulham Road, London SW3 6JB, UK. charlotte.dodson@chem.ox.ac.uk
authors.forename: Charlotte A
authors.initials: CA
authors.lastname: Dodson
authors.forename: Sharon
authors.initials: S
authors.lastname: Yeoh
authors.forename: Tamanna
authors.initials: T
authors.lastname: Haq
authors.forename: Richard
authors.initials: R
authors.lastname: Bayliss
mesh_terms: D064096:Aurora Kinase A / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015153:Blotting, Western; D064447:Checkpoint Kinase 2 / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D013058:Mass Spectrometry; D008954:Models, Biological; D008958:Models, Molecular; D009154:Mutation; D000072379:NIMA-Related Kinases; D010455:Peptides / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D011494:Protein Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D010455:Peptides; D013912:Threonine; D011494:Protein Kinases; C490219:PLK4 protein, human; D064447:Checkpoint Kinase 2; D064096:Aurora Kinase A; C512685:NEK7 protein, human; C462119:NEK9 protein, human; D000072379:NIMA-Related Kinases; D017346:Protein Serine-Threonine Kinases